Introduction
This field protocol takes you through the steps of testing fresh or marine water for fecal contamination using qPCR specific for Enterococcus faecalis (universal stool marker) and HF183 Bacteriodes 16S rRNA (human stool specific). These markers were selected based on extensive water contamination research carried out by the CDC, which you can read about here, and are available as a multiplexed QPCR assay (together with an internal positive control) sold by Biomeme Inc. as the BioPoo assay. This protocol uses the Biomeme portable qPCR platform and consumables, which allows the entire assay to be carried out in the field, without electricity or access to standard lab equipment.
We will be using the Biomeme eDNA 'BioPoo' Enterococcus assay. Each tube contains a triplex assay that detects a general fecal marker, Enterococcus faecalis (green channel), a human-specific fecal marker, HF183 Bacteriodes 16S rRNA (amber channel), and an Internal positive control or IPC (red channel).
You can view a video on adding loading purified sample to Go-Strips here and then placing Go-Strips into your Franklin thermocycler here.
Safety
- Follow universal lab safety precautions: wear a lab coat and gloves when working in the lab, no open-toed shoes, no eating and drinking.
Reagents
Reagent | Vendor | Catalog | Storage |
BioPoo Enterococcus Panel RT-PCR Go-Strips Test | Biomeme | 3000533 | Room Temp |
Molecular-Grade Water | Sigma | W4502-1L | Room Temp |
Equipment and Consumables
Item | Vendor | Catalog |
Biomeme | 1000003 | |
iPhone with Biomeme GO app loaded | Biomeme | Via App Store |
MySpin 12 mini centrifuge (max 12,500 rpm - 9,800 rcf) | Thermo Scientific | 75004081 |
P20 Pipet and Filter Tips | NA | NA |
Protocol
qPCR Reaction Set-Up
- Remove three Go-Strips (9 tubes) from the pack and set them in your PCR rack with the tube connections facing away from you and the tab on the foil seal to the left. Label each tube with the sample number.
- Carefully (and with a gloved hand) pull the seal off of each Go-Strip.
- Working with one DNA extract at a time, use a pipet to transfer 20 ul of DNA to a single tube in a Go-Strip. Pipet up and down slowly 10X to thoroughly resuspend and mix lyophilized reagents.
Raise the Go-Strip to eye level to make it easy to see what you're doing. Avoid creating bubbles, particularly at the bottom of the tube. Change tips between tubes to avoid assay cross-contamination.
- Once DNA has been added to all three tubes in a Go-Strip, add rubber caps with the notches in the back.
- Repeat this process with the remaining two Go-Strips.
- You should now have three Go-Strips (9 tubes) that contain your 9 DNA extracts that are well mixed and capped.
- Add the Go-Strips to the Biomeme instrument with the notches in the back. Close the lid (this pushes the void-filling caps deep into the tubes), pair your phone with the device, and follow the instructions on the phone for setting up your run.
Login to our lab's Biomeme account on the app using the username beiting@vet.upenn.edu and the password Biomeme1234! (case sensitive). Select 'hostmicrobe-three9' for the team.
When using your phone to set up the QPCR run, if the QR code on the Go-Strip package isn't scanning properly, try this fix. After you've logged in as described above, from the app home screen:
1. Click the settings gear icon (top right).
2. Select "Settings"
3. Scroll down and select "Advanced Network Settings"
4. Select "Sync Protocols"
- Interpret results (see below) and record them in the GERA Metadata and QPCR Results document.
See the Biomeme Reattach and Recover Guide if you accidentally disconnect from the app or instrument during the run.
Interpreting Your Results
Perhaps the most important thing to remember when interpreting your results is that, since the BioPoo assay is based on the detection of DNA, a positive result only means that the target was detected, not that there are live organisms present. For this reason, no conclusions about water potability or safety of recreational water should be based on results from the Biopoo assay. If you’re interested in EPA-approved assays for microbial detection in water, you may want to take a look at the ‘EPA Method 1600’, or Enterolert and Colilert assays from IDEXX.
- A good strategy is to run all 9 of your samples, take time to review the results on your phone, then decide which, if any, samples need to be rerun.
- Because the IPC is so bright, it is best to view each assay 'color' separately in the software (simply touch to toggle colors on/off), otherwise, the IPC signal (red) can overwhelm and dwarf the Entero (green) or HF183 (amber) signals on the graph.
- When viewing your data, be sure to also toggle between 'baseline' and 'raw' views. If you see a weak signal and are wondering if it is real, you should see it in both views. On the other hand, if a particular sample only appears positive in baseline view, but not raw, then it is probably not real.
Possible Assay Outcomes
Outcome | Likelyhood | Enterococcus (Green) | HF183 (Amber) | IPC (Red) | Interpretation |
1 | Most Likely | X | Water sample is NOT contaminated with human or animal feces. | ||
2 | Possible | X | X | X | Water sample is contaminated with human feces. |
3 | Possible | X | X | Water sample is contaminated with feces, but likely from animals. | |
4 | Possible | IPC failed. Possilbe PCR inhibitors. Rerun sample with lower input (see below for more info). | |||
5 | Unlikely | X | X | This would be an unusual result and may require a rerun. | |
6 | Unlikely | X | IPC failed. Rerun if Green/Amber curves do not look good (see below for more info). | ||
7 | Unlikely | X | IPC failed. Rerun if Green/Amber curves do not look good (see below for more info). | ||
8 | Unlikely | X | X | IPC failed. Rerun if Green/Amber curves do not look good (see below for more info). |
Troubleshooting Failed Runs
- The IPC will fail when there is a strong Enterococcus or HF183 signal because of competition of available reagents. If the Enterococcus and/or HF183 curves look good (see example below), there’s no need to rerun.
- If all three signals (IPC, Enterococcus AND human, see below) fail for one or a few samples on a run, it’s possible that natural inhibitors present in the sample may have made it through extraction and are blocking your PCR reaction. This often happens if a lot of biomass was captured on the filter funnel and/or the syringe filter tip begins to clog during DNA extraction. To address this possibility, repeat the assay with 4 µl of DNA instead of 20 µl of DNA. This 5-fold reduction in input often dilutes the inhibitors enough that the reaction proceeds normally. If you still get poor results, try using 2 µl or 1 µl of DNA as input. Keep the total reaction volume at 20 ul with molecular-grade water.
- If all samples from a run fail (see below), power cycle the Biomeme instrument AND phone app and repeat the assay. Be sure to thoroughly mix the DNA in the reagent tubes before loading.
